Ketogenic diet enhances the effects of oxycodone in mice

Opioids have been used to manage pain for thousands of years, but they have significant potential for abuse. Prescription opioids, like oxycodone, are associated with 32% of overdoses, that have reached a total of 75,673 deaths in 2021. A major challenge is maximizing their therapeutic potential while minimizing the negative side effects including opioid use disorder (OUD). The Ketogenic Diet (KD) has been reported to reduce pain and decrease the severity of alcohol use disorder, yet its effects on oxycodone responses remain unknown. KD mice displayed increased oxycodone-induced locomotor activity and enhanced antinociceptive effects of oxycodone, suggesting a dietary effect on opiate sensitivity. Male KD mice exposed to chronic oxycodone exhibited increased naloxone-induced jumps, suggesting a sex-specific effect of diet on opioid withdrawal. Consistent with this, male KD mice self-administered less oxycodone while female KD mice did not differ from controls. Finally, no effect of KD on motivation to obtain oxycodone was observed during a progressive ratio schedule. These data suggest sex-biased effects of KD on responses to opioids that should be considered and potentially leveraged in both clinical pain management and treatment of OUD.

Drugs. Saccharin (Acros Organics #149005000) was diluted to several concentrations (the highest being 0.1%) in tap water. Oxycodone HCl was supplied by the NIDA drug sharing program. Oxycodone was diluted at various concentrations in either tap water for self-administration experiments, or sterile saline for injections and osmotic minipump loading. Naloxone Hydrochloride was purchased from Spectrum Chemical (#N1231), dissolved in sterile saline, and injected at 1 mg/kg intraperitoneal (i.p.) immediately prior to assessing jumping behavior. Lidocaine (Covetrus, North America VINB-0024-6800) was injected locally at 4 mg/kg subcutaneously (s.c.) prior to incision for minipump implantation. Carprofen (Zoetis) was injected i.p. at 5 mg/kg prior to incision for minipump implantation, and again at 24 and 48 h after surgery.
Locomotor activity. Med Associates locomotor chambers, which utilize infrared beams, and Med PC V software were used to assess locomotor activity. All mice were habituated to Med Associates locomotor chambers for 90 min, during which they all received a saline injection i.p. at 60 min. Following the 90 min habituation period, mice then received an acute 2 mg/kg oxycodone injection i.p., and activity was measured over an additional 90 min in 5 min bins. Two consecutive beam breaks were required to register as locomotor activity.
Hot plate assay. Mice that were previously used for locomotor activity were assessed for nociception, with a minimum of two days between tests. The hot plate assay was conducted using a Series 8 hot plate from IITC Life Science held at a constant 52 °C. A 40 s cutoff time was implemented to avoid tissue damage. Nociceptive measures included paw flick, paw lick, or jumps. Each mouse was immediately removed from the hot plate after the first incidence of nociception or 40 s, whichever came first. Baseline nociception was tested first. Then 2 mg/ kg oxycodone was delivered i.p. and nociception was tested at 10, 20, 30, and 45 min post-oxycodone. Precipitated withdrawal. Osmotic minipumps (Alzet #1003D) were used to chronically deliver oxycodone over three days. Mice were placed on their respective diets ad libitum for four days prior to osmotic minipump surgeries. The day before surgery, bodyweights were assessed, and minipumps were custom loaded to deliver 130 mg/kg/day. Ketone levels were measured prior to surgery. Surgeries were performed under aseptic conditions. Briefly, mice were injected with carprofen, anesthetized with isoflurane, and shaved at the incision site on their backside lateral from their spine. The exposed skin was cleaned with betadine, lidocaine was injected locally, and a small incision was made with scissors. A subcutaneous pocket was made and the minipump was inserted. Wound clips were used to close the incision site and triple antibiotic cream was applied. Animal welfare was monitored daily following surgery. Three days after surgery, mice were injected with 1 mg/kg naloxone i.p. and placed in an empty static mouse bucket with a high lid. Jumping behavior was chosen as our measure of withdrawal as this has consistently been the most objective and quantifiable outcome observed in our lab. Jumps were quantified using Med Associates locomotor chambers, with the beam detectors raised approximately six inches off the ground, high enough to require all four paws leaving the cage floor, and to not detect rearing. A jump was counted when an animal broke a beam, with a 0.5 s timeout period to allow for the mouse to land. Jumps were quantified over 20 min. www.nature.com/scientificreports/ Oxycodone self-administration and progressive ratio. All mice were maintained on standard chow and food restricted to 85-90% of their bodyweight. Med Associates operant boxes were used, which were equipped with three nosepoke ports on one side, a magazine for reward retrieval on the opposite wall, cue lights in the active nosepoke port and magazine, and a syringe pump for delivery of liquid rewards. Five ml syringes were used, and pump activation time was set to deliver 20 µl of liquid into the magazine per reward earned. The operant sequence of events started with activation of the nosepoke port as indicated by illumination of its cue light. Upon a successful nosepoke port entry, the nosepoke cue light was turned off, a reward was immediately delivered and a cue light in the magazine was turned on. Upon successful magazine entry by the mouse, the magazine cue light was turned off, and after a 5 s delay, the appropriate nosepoke port was made active again, as indicated by its cue light. Self-administration sessions were 3 h long. Mice were initially trained to self-administer 0.1% saccharin solution in water on a fixed ratio 1 (FR1) schedule, in which each active nosepoke resulted in the delivery of saccharin reward, which then was increased to an FR3 schedule (3 nosepokes per reward). After several days of FR3 0.1% saccharin, oxycodone was gradually introduced over several days, from 0.05 mg/ml to 0.3 mg/ml. After four days at 0.1% saccharin 0.3 mg/ml oxycodone, the saccharin was fully faded out over four days. After six additional days of 3 h sessions at FR3 with unsweetened oxycodone, mice were assigned to their dietary groups (KD or Chow), counterbalanced based on their oxycodone consumption. Self-administration sessions and food restriction were maintained throughout the dietary transition. Magazines were checked at the end of each session to verify rewards had been consumed. Mice were then tested on an exponentially increasing progressive ratio schedule [26][27][28] . The session was conducted over 6 h, with drinking water provided in the operant box to eliminate thirst as a confound for liquid seeking. No cutoff time was implemented in the program. The nosepoke requirement schedule (where j = 0.2) for the first 14 rewards is as follows: 1, 2, 4, 6,9,12,15,20,25,32,40, 50, 62, 77.

Progression of ketosis and the effects of KD on bodyweight.
To confirm efficacy of the KD regimen in inducing a ketogenic state, ketone levels were measured in male and female mice that were maintained under ad libitum conditions on either KD or Chow. Ketone levels were measured prior to diet initiation (day 1), and on days 4 and 7 post-diet induction, which revealed a significant increase in β-hydroxybutyrate levels by day 4 for both sexes ( Fig. 1A and B). The male mice consistently lost weight on the KD relative to Chow males over the course of eight days (Fig. 1C). In contrast, the female mice displayed no differences in bodyweight between KD and Chow groups over the course of eight days (Fig. 1D).
Locomotor activity. To assess general changes in sensitivity to oxycodone due to the KD, we assessed locomotor activity in separate cohorts of male and female mice before and after an acute oxycodone challenge. The male cohort included a Pair-fed Chow group to account for the potential contribution of bodyweight loss on oxycodone sensitivity and response. KD, Chow, and Pair-fed Chow groups were placed on their respective diets for a minimum of four days. β-hydroxybutyrate levels (mM) were assessed in males prior to locomotor testing: Locomotor activity was recorded during each phase (baseline, saline, and oxycodone). Compared to Chow, KD male mice showed an enhanced locomotor response to oxycodone when measured repeatedly over time as well as the cumulative activity within the oxycodone phase ( Fig. 2B, C).
Unlike male mice, female mice showed no differences in the relative change in bodyweight percentage from the start of dietary intervention to the time of locomotor testing (Fig. 2D). Similar to the males, female KD mice displayed an enhanced locomotor response to oxycodone compared to Chow as evaluated across time bins, as well as cumulative activity per phase (Fig. 2E, F). The female Chow mice displayed slightly higher activity than KD mice during the Baseline phase.
Hot plate. A hot plate assay was used to assess changes in the antinociceptive effects of oxycodone in males and female mice. Time to nociceptive behavior was recorded at five time points: baseline (BL) prior to 2 mg/kg oxycodone injection, and 10, 20, 30, and 45 min after injection. No difference between male dietary groups was observed at baseline (BL), however latency to show discomfort was significantly increased in the KD mice with a main effect of diet after oxycodone injection (Fig. 3A). Similar to the males, the female mice had no difference at BL between groups, however latency was significantly increased in the KD mice with a main effect of diet after oxycodone (Fig. 3B). There were no differences in bodyweight loss among the Pair-fed Chow vs KD males, and with Chow vs KD females (Fig. 3C).

Precipitated withdrawal.
To assess the effects of KD on withdrawal symptoms, we measured jumping behavior after inducing withdrawals with naloxone. After mice had completed three days of continuous oxycodone delivery via osmotic minipumps, jumps were quantified over twenty minutes immediately after naloxone injection (1 mg/kg i.p.). Male mice in the KD group had significantly more jumps than Chow mice (Fig. 4A). No difference was observed with female mice comparing KD vs Chow (Fig. 4B). www.nature.com/scientificreports/ Oxycodone self-administration and progressive ratio tests. Male and female mice were trained to operantly self-administer oral oxycodone while maintained on standard chow. After their Day 6 self-administration session, the dietary intervention experiment was started by replacing the standard chow with ketogenic diet for the KD group. We observed a significant reduction in oxycodone consumed by KD males (Fig. 5A), and a trend reduction by KD females (Fig. 5B). These mice were then tested for motivation to acquire oxycodone using a progressive ratio paradigm. Male and female mice showed no difference in rewards earned between KD and Chow groups over the test session ( Fig. 5C and D).

Discussion
These data suggest that KD modulates opioid responses in mice in a sex-specific manner (Table 1). While KD induced substantial weight loss in male mice, this was not the case in female mice despite effective induction of ketosis in both sexes. Strikingly, we found that KD augmented antinociceptive and locomotor effects of oxycodone in both sexes. While KD exacerbated oxycodone withdrawal in males, this effect was absent in female mice. www.nature.com/scientificreports/ KD also reduced oxycodone self-administration, especially in early sessions following KD diet administration, and with a more pronounced effect in male than female mice. More broadly, these data serve as an example of the importance of nutritional and metabolic state for both therapeutic use of opiates for pain management, as well as treatment of OUD. While both male and female mice on KD rapidly develop ketosis, we observed sex differences in bodyweight loss in KD male mice only. This persisted throughout locomotor and hot plate testing. This sex-specific  www.nature.com/scientificreports/ bodyweight response to the ketogenic diet in mice has not been reported in the literature, however it is consistent with clinical data evaluating patients with obesity on a ketogenic diet, where it was reported that male subjects lost more weight than females 29 . Oxycodone-induced locomotor activity was tested to screen for general changes in opioid sensitivity on the ketogenic diet. Both male and female KD mice displayed robust increases in activity after oxycodone treatment compared to Chow mice, indicating that the ketogenic diet may enhance the effects of the opioid. Interestingly, the female KD mice displayed significantly less activity during baseline and saline phases than Chow. This reversal from lower to higher oxycodone-induced activity strengthens the finding that the dietary manipulation enhanced their opioid sensitivity.
Since it is known that food restriction can enhance behavioral and physiological sensitivity to drugs of abuse 30 , we generated a Pair-fed Chow group matched to the KD group's bodyweight loss in male mice. β-hydroxybutyrate levels were significantly different across all three male groups prior to locomotor activity testing, which was expected as food restriction can increase ketone levels 31,32 . However, the KD diet produced much higher β-hydroxybutyrate levels than the Pair-fed Chow, and even more importantly, the Pair-fed Chow mice did not show the enhanced locomotor and antinociceptive responses to oxycodone, suggesting that these effects are primarily driven by the ketogenic diet, and not by the bodyweight loss.
We also evaluated effects of the KD on modifying sensitivity to the antinociceptive effects of oxycodone using the hot plate assay. Both male and female KD mice displayed significantly enhanced latency after oxycodone compared to Chow, suggesting that the ketogenic diet may enhance the antinociceptive effects of oxycodone. These data support a potential role for a ketogenic diet as an adjunct with the therapeutic use of opiates for pain management in a clinical setting. In line, as the ketogenic diet has shown efficacy in reducing pain in animals and humans 20,22,33 , it has recently been recommended as a potential adjunct to a comprehensive pain management plan 22 . These data provide more scientific evidence for those recommendations and need clinical validation.
We evaluated KD effects on physical withdrawal symptoms after chronic oxycodone delivery via minipump. In this case, dietary manipulation occurred prior to oxycodone exposure, and withdrawal was evaluated after ketosis had developed. Interestingly, male KD mice had increased jumping behavior after naloxone treatment, suggesting increased sensitivity to withdrawal symptoms, whereas the female KD mice did not show these effects. Again, these data are consistent with an increased sensitivity to oxycodone caused by the KD. These data, along with the sex differences in bodyweight loss, suggest sexual dimorphism of the KD that need further exploration. No studies have investigated directly the influence of sexual dimorphisms of diet on OUD, but several have determined other factors driving OUD responses, such as exercise, weight gain, and also the development of OUD [34][35][36] .
We also evaluated effects of KD on self-administration of oral oxycodone. After dietary intervention, male KD mice showed a significant reduction in oral self-administration compared to Chow, and female KD mice showed a trend reduction as well. As only one concentration of oxycodone was tested in self-administration and progressive ratio paradigms, future studies evaluating additional concentrations may be of value in confirming translational relevancy. These data may have translational significance by showing that a dietary intervention can impact ongoing opiate consumption. However, clinical studies are necessary to evaluate whether or not this reduction in consumption would have any potential effects on withdrawal symptoms, which in turn might contribute to successful treatment of OUD. Follow up studies are needed to confirm potential efficacy, as well as to test for KD interactions with OUD medications (i.e., methadone, buprenorphine, naltrexone). Notably, neither male nor female KD mice displayed a significant difference in motivation on the progressive ratio schedule, suggesting limited effects of the KD on motivation during intake.
Overall, we show a modulatory role for KD in the response to opioids and opioid withdrawal symptoms. While both sexes experienced improved analgesic effects during opiate use for pain relief, a KD was only efficacious in reducing opiate consumption in male mice, whereas the females exhibited a strong trend in reduction. Meanwhile, withdrawal was selectively enhanced by KD in male, not female, mice. This sex-dependent response profile could have implications for clinical implementation strategies, but direct studies in human subjects are needed. OUD is a complex disorder with a multitude of variables that impact treatment outcomes. Sex differences may be a major factor influencing treatment outcome among OUD patients, with some studies showing that women progress to more severe OUD faster than men, and have higher withdrawal scores, whereas other studies show that men are less likely to remain in treatment 37 . The present findings support the need for an overall Table 1. Summary of findings. Up arrows indicate an increase, down arrows indicate a decrease, bidirectional horizontal arrows indicate no change. N/A indicates that group wasn't included. The effect size (degree of significance) for a given readout is indicated by the number of arrows.